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Nutritional, Sensory and Phytochemical Evaluation of High Protein Granola from Quinoa and Amaranth

V
Vishal P. Raut1
0009-0001-4747-9704
M
Manmath D. Sontakke1,*
0000-0003-1132-8864
S
Supriya M. Kamble1
0009-0009-5038-3293
S
Shivam G. Salunke2
1Department of Food Processing Technology, Institute of Biosciences and Technology, MGM University, Chhatrapati Sambhajinagar-431 001, Maharashtra, India.
2Sau. K.S.K. (Kaku) College of Food Technology, Beed-431 122, Maharashtra, India.

ABSTRACT

Background: The utilization of and plant-derived components is on the rise in the formulation of functional foods that improved nutritional and antioxidant characteristics.

Methods: Formulations for granola were made using varying level of amaranth, quinoa, foxnut, whey protein isolate, pumpkin seeds and honey. Proximate composition, sensory attributes, antioxidant activity, total phenolic content and, total flavonoid content and color analyses of the samples were determined by the standard analytical methods.

Result: The granola prepared in this study showed better nutritional and functional qualities than control sample. The protein content of the formulations with whey protein isolate with amaranth was significantly superior, F2 showed 31.2% protein conten and F5 showed the highest energy value (394 kcal). In addition, improved the levels of dietary fibre and minerals like calcium, iron, magnesium, zinc and phosphorus. A color analysis revealed that there were differences between formulations, the ΔE ranged from 1.73 to 7.15. Quinoa, amaranth, pumpkin seeds, foxnut and honey had a significant improvement of antioxidant activity, total phenolic content and the amount of flavonoids. In the formulations, F3 showed the best bioactive and antioxidant effects along with the best mineral composition.

INTRODUCTION

Growing consumer awareness of the relationship between diet and health has increased demand for functional foods that provide benefits beyond basic nutrition. Breakfast cereals, especially granola are popular due to their convenience and nutritional values. However many commercial granola are high in sugar and relatively low in protein, creating need for the healthier alternatives (Granato et al., 2020). Commercial cereals are often a source of poor dietary quality and “empty calories” due to their high sugar, salt and saturated fat content and comparatively low dietary fiber, protein and key micronutrient content (Sharma et al., 2020; Singh and Singh, 2019).
       
The possible health benefits of pseudo cereals unveil that for the preparation of functional food development (Joshi et al., 2019). Examples of pseudo-cereals that have high nutritional quality are amaranth, buckwheat and quinoa. The main reason for the use of quinoa today is its high protein % and quality - the balanced amino acid spectrum with a high lysine and methionine content (Abugoch et al., 2009; Wright et al., 2002;). High in fiber and minerals like calcium and iron (Ando et al., 2002). It is also rich in antioxidants, such as polyphenols (Hirose et al., 2010; Repo-Carrasco-Valencia et al., 2011). Also high protein content in amaranth makes it ideal for use in the preparation of healthful food (gluten-free) for people with gluten hypersensitivity (Capriles et al., 2014). Similarly, Foxnut (Euryale ferox) and pumpkin seeds are rich sources of natural protein (25-37%) and oil (37-45%) antioxidants, minerals and nutrients that contribute to overall heath (Mishra et al., 2022; Kaur et al., 2018; Chauhan et al., 2025). Whey protein isolate (WPI), containing more than 90% protein and all essential amino acids, is widely used to enhance the nutritional quality of food products complementing the plant proteins present in Pseudocereals and seeds (Smithers, 2015). The granola is sweetened with honey and dried fruits/fruit pastes, which are used to increase the energy content of the granola. The honey is natural sweetener, having therapeutic characteristics as well as binder for producing (Deshmukh et al., 2017).
       
Previous studies have reported the successful incorporation of Pseudocereals and seeds into ceareal- based product, improving their nutritional and functional properties (Rocchetti et al., 2021). However, limited research has investigated the combined use of amaranth, quinoa, Foxnut, pumpkin seeds, honey and whey protein isolate in granola formulations and understanding the complex interactions among ingredients. The combination of these ingredients is expected to create a synergistic effect by improving protein quality, minerals density, antioxidants potential, texture, flavour and overall consumer acceptability. Furthermore, information regarding the physicochemical, textural and evaluate its nutritional, physicochemical, textural and sensory properties.
       
The research aims to develop gluten-free and nutritious rich Amaranth, Quinoa Granola with the inclusion of Foxnut, Pumpkin seeds with its deliciousness and evaluate its nutritional, physicochemical, textural and sensory properties.

MATERIALS AND METHODS

Raw material required for the formulation are Amaranth grain (Amaranthus hypochondriacus), quinoa flour (Chenopodium quinoa), foxnut (Euryale ferox), pumpkin seeds (Cucurbita pepo), honey and whey protein isolate (WPI) of functional granola are procured from local market of Chhatrapati Sambhajinagar, Maharashtra, India. Whey protein isolate (90% protein) was purchased from a commercial manufacturer. Honey was used as the natural sweetener and binder because of its nutritional value and adhesive properties. All the analysis was carried out at Institute of Biosciences and Technology, MGM University Chhatrapati Sambhajinagar, Maharashtra in the year 2024 to 2025.
 
Formulation of functional granola
 
Five alternative formulations were built to find the most acceptable sensory properties by varying the amount of amaranth, quinoa and whey protein (Table 1) expressed in % of the total. The 25% honey was uniformly added as a binding agent due to benefits of honey as a texture, cohesion and sensory improver in granola and cereal based product (Sharma and Gujral, 2020; Deshmukh et al., 2017). The proportion of roasted pumpkin seeds and Foxnut was maintained constant in all five formulations. The prepared sample of granola is shown in Fig 1.

Table 1: Formulation for standardization of functional granola.



Fig 1: Process of formulated functional granola. (Reference: Ahada et al., 2025).


 
Proximate analysis
 
Proximate composition of formulated granola was determined in triplicate (n=3) using standard methods of the AOAC International. Moisture content was determined by the Hot air oven method (AOAC method 925.10), crude fat by Soxhlet extraction, model-soxtron sox-2, crude protein by Kjeldahl method (Kjeltron KGIDB4M, India) using a nitrogen conversion factor of 6.25 (AOAC method 979.09) and total ash by incineration in muffle furnace (AOAC method 923.03) Muffle furnace, Biotechnics India). The total carbohydrate content was calculated by the difference method according to AOAC procedure using the following equation:

Carbohydrates (%) =
100 - [Moisture (%) + Protein (%) + Fat (%) + Ash (%)]
 
Sensory analysis
 
The sensory evaluation was conducted by 15 semi-trained panellists (aged 22-45 years) familiar with cereals based products. Samples were coded with random three-digit number and served in a randomized order under controlled laboratory conditions. Color and appearance, aroma, taste, body and texture and overall acceptability were evaluated using a nine-point hedonic scale (9= like extremely; 1= dislike extremely). The panel size was considered adequate for preliminary acceptability testing of newly developed food products. Sensory score were analysed using one-way ANOVA followed by Duncan’s multiple range test (DMRT) at p < 0.05 (Lawless et al., 2010).
 
Shelf-life study: Moisture, pH, acidity and peroxide value
 
Shelf life of the formulated functional granola was evaluated following the method of Iuliano et al., (2019) did, but with slight modifications. Granola samples were packed in laminated aluminium foil polyethylene pouches and stored at room temperature (30±2oC) under ambient relative humidity conditions for 28 days. Analyses were performed in triplicate at 0, 7, 14, 22 and 28 days of storage. Moisture content (AOAC, 2019), pH, titratable acidity, peroxide value and free fatty acid (FFA) content were determined to access product stability. The 28-day storage period was selected as a preliminary shelf-life assessment to monitor physicochemical changes and oxidative stability of developed granola under normal conditions.
 
Antioxidant activity, total phenolic content and total flavonoid content
 
The antioxidant activity, total phenolic content (TPC) and total flavonoid content (TFC) of the formulated granola were determined in triplicate (n=3). Briefly, 1 g of finely ground granola was extracted with 10 mL of methanol (1:10 w/v), vortexes for 5 min and centrifuged at 1400 rpm for 30 min. The supernatant was collected and used for further analyses. Antioxident activity was evaluated using the DPPH radical scavenging assay described by Dubey et al. (2023) and absorbance was measures at 517 nm using a UV-Vis spctrophotmeter [(UV_!*) Shimadzu Corpration, Japan]. The results were expressed as percentage inhibition using the equation:

 
Where
A0 = Absorbance of the control.
As = Absorbance of the sample.
       
The TPC was determined by Folin-Ciocalteu method using gallic acid as the standard and expressed as mg GAE/100 g sample (Pérez et al., 2023). TFC was determined using the aluminium chloride colorimetric method with quercetin as the standard and expresses as mg QE/100 g sample (Shraim et al., 2021).
 
Mineral analysis
 
Calcium, iron, magnesium, zinc and phosphorus content were determined in triplicate according to AOAC (2019). Granola samples were dry-ash at 550oC and the ash was dissolved in dilute hydrochloric acid, filtered and diluted to a known volume. Calcium, iron, magnesium and zinc were quantified using Atomic Absorption Spectrophotometry (AAS) with certified standard solution for calibration, while phosphorus was determined colorimetrically using a standard phosphate calibration curve. Quality control was ensured through reagent blanks, calibration verification standards and duplicate analyses. Mineral concentration was expressed as mg/100 g sample.
 
Color analysis
 
The color characteristics of the formulated granola samples were measured using a Hunter Lab MiniScan XE Plus colorimeter. Color values were recorded as L* (lightness), a* (redness/greenness) and b* (yellowness/blueness). The overall color difference (ΔE) between samples was calculated using the CIE Lab* color difference equation (Pathare et al., 2013):.
 
ΔE = √[(ΔL)2 + (Δa)2 + (Δb*)2]**

Where 
ΔL*, Δa* and Δb* = Differences in lightness, redness/greenness and yellowness/blueness, respectively. Measurements were performed in triplicate and results were expressed as mean ± standard deviation.
 
Statistical analysis

All experiments were performed in triplicate (n=3) and results were expressed as mean ± standard deviation. Data were analysed using Minitab 20 statistical software. A completely randomized design (CRD) was employed and differences among the six granola formulations (control and five treatment formulation) were evaluated using one-way analysis of variance (ANOVA). Mean comparisons were performed using tukey’s H Honestly Significant Difference (HSD) test at a 95% confidence level (p<0.05). Sensory and storage study data were analysed using the same statistical approach to determine significant differences among formulations and storage periods. 

RESULTS AND DISCUSSION

The proximate composition of the formulated granola is presented in Table 2. Protein content increased significantly from 11.8±0.24% in the control to 31.2 ± 0.38% in F2 (p<0.05), mainly due to the addition of whey protein isolate, while amaranth and quinoa provide complementary pant protein (Boire et al., 2025). The slight increase in fat content (5.7-6.8%) was attributed to the healthy unsaturated fats present in pumpkin seeds and Pseudocereals. Crude fibre increased with the incorporation of amaranth , quinoa and Foxnut, with F3 showing the highest value (6.2±0.10%), owing to their naturally high dietary fibre content (Jan et al., 2023) the higher ash content observed in F5 reflected the greater mineral contribution from Pseudocereals and pumpkin seeds, which are rich in calcium, magnesium, iron and zinc (Anuradha et al., 2023; Olawuni et al., 2024).Consequently, carbohydrate content decrease because it was replaced by protein – and fibre-rich ingredient, while the energy value (372-394 kcal/100 g) remained comparable among formulations. These results demonstrate that combined use of Pseudocereals, pumpkin seeds and whey protein isolate enhanced the nutritional quality of granola by improving protein, fibre and mineral contents without substantially increasing energy value.

Table 2: Nutritional properties of functional granola.


 
Total phenolic content, antioxidant activity, total flavonoid content
 
The antioxidant activity, total phenolic content (TPC) and total flavonoid content (TFC) of the granola formulations are presented in Table 3. Formulations enriched with higher proportions of amaranth and quinoa (F3 and F4) exhibited significantly higher antioxidant activity, TPC and TFC due to the abundance of phenolic acid and flavonoids, including quercetin and kampferol, natural present in these Pseudocereals (Alvarez-Jubete et al., 2010).however, not all formulations showed similar improvements. The comparatively lower values observed in F1, F2 and F5 % may be attributed to a dilution effect caused by a higher proportion of ingredients with lower phenolic concentration, as well as possible interactions between protein and phenolic compounds that reduce the extractability of bioactive compounds during analysis (Tang and Tsao, 2017; Zhu, 2025). These findings indicate that the antioxidants potential of granola depends on the composition and interactions among ingredients rather than the addition of functional ingredients alone.

Table 3: Phytochemical and antioxidant properties of functional granola.


       
The formulated granola also showed improved mineral composition. Higher calcium, iron, magnesium, zinc and phosphorus content were mainly associated with the incorporation of quinoa, amaranth, pumpkin seeds and whey protein isolate, all of which are naturally rich in essential minerals (Alvarez-Jubete et al., 2010; Aremu et al., 2022:). This demonstrates that combining Pseudocereals with protein-rich ingredients enhances both the bioactive and minerals profiles of the developed granola.
 
Color analysis  
 
The color characteristics of the formulated granola are presented in Table 4. The L * values (56.41±0.33-62.68± 0.24) indicates differences in lightness among the formulations, while positive a* values (6.71±0.08-7.72±0.09) and b* values (24.08±0.21-27.55±0.19) confirmed the characteristic reddish-yellow appearance of the granola. These variations were mainly attributed to differences in ingredient composition, particularly the levels of honey, pumpkin seeds, amaranth and quinoa as well as browning reactions during roasting (Szydłowska et al., 2022). The ΔE values indicated perceptible color differences among formulations, suggesting that ingredient substitution produced visually distinguishable products that may influence consumer perception and product acceptability. Overall, the color changes reflected both formulations differences and processing conditions.

Table 4: Color analysis of functional granola.


 
Sensory evaluation of functional granola
 
The sensory scores (Table 5) showed that all formulations were well accepted, with F5 receiving the highest overall acceptability score (H≈9), followed by F1 and F2. The superior acceptability of F5 may be attributed to the balanced combination of amaranth, quinoa, Foxnut, pumpkin seeds, honey and whey protein isolate, which provided a desirable flavour, crisp texture, attractive color and pleasant mouth feel. In contrast, formulations containing higher levels of amaranth (F3 and F4) showed comparatively lower scores, likely due to its characteristic earthy flavour and denser texture, which may have reduced consumer preference (Sharma and Kaur, 2021). The consistent scores obtained from the semi-trained panel indicate good agreements among panellists and support the reliability of the sensory evaluations.

Table 5: Sensory evaluation of functional granola.


 
Change in moisture content, pH, acidity, peroxide value
 
Granola formulations F1-F5 showed consistent moisture stability over the 28-day storage period ranging from about 4.8% to 6.2% moisture. In the samples F2 had a slight decrease from the high moisture content at the beginning and F3 and F4 showed more stable moisture content.
       
The storage stability of the formulated granola was evaluated over 28 days by monitoring moisture content, pH, titratable acidity and peroxide value (Fig 2). Moisture content showed only a slight change (4.8-6.2%) during storage, indicating that the aluminium foil- polyethylene packaging effectively minimized moisture transfer and maintained produt quality (Fellows, 2009; Robertson, 2016). The pH decreased significantly (p<0.05) from 6.5-6.7 at day 0 to 5.6–6.1 on day 28, reflecting the formation of free fatty acids and other oxidation products during storage (Choe and min, 2006; Zhang et al., 2019).

Fig 2: Effect of storage on moisture and pH of functional granola.


       
Titratable acidity increases significantly p < 0.05) in all formulations, from 0.1-0.2% at day 0 to 0.4–0.6% after 28 days. F2 exhibited the highest acidity (0.6%), followed by F3 (0.5%), whereas F1, F4 and F5 reached 0.4%, (showed in Fig 3) suggesting comparatively lower hydrolytic deterioration (Gumus and Decker, 2021). Similarly, peroxide value increased (Fig 3) from 1.8-2.3 to 6.2-6.9 meq O2/kg during storage, indicating progressive lipid oxidation. F4 recorded the highest peroxide value (6.9 meq O‚ /kg), while F3 showed the lowest (6.2 meq O2/kg), demonstrating relatively better oxidative stability (Gumus and Decker, 2021; Shahidi and Zhong, 2015). Although significant physicochemical changes occurred during storage (p< 0.05), all formulations maintained acceptable quality throughout the 28-days storage period under ambient conditions.

Fig 3: Effect of storage on Acidity and peroxide value of functional granola.


       
The higher total phenolic and flavonoid content in F3 contributed to better oxidative stability, as reflected by its lower peroxide value (6.2 meq O2/kg) after 28 days. Phenolic compounds act as natural antioxidants by scavenging free radicals and delaying lipid oxidation (Shahidi and Zhong, 2015). In contrast, F3 showed the highest peroxide value (6.9 meq O2 /kg), indicating that oxidative stability depends not only on antioxidant content but also on ingredient composition and interaction among bioactive compounds.

CONCLUSION

Overall, the developed Pseudocereals based granola exhibited improved nutritional guilty, with higher protein, fibre, mineral and bioactive compound content while maintaining a comparable energy value. The optimized formulation demonstrated colour, good sensory acceptability and satisfactory oxidative stability, indicating that ingredient composition and interaction significantly influenced product quality. These findings suggest that the combined used of Pseudocereals, pumpkin seeds and whey isolate is an effective approach for developing a nutritious and functional granola with enhanced health-promising properties.

ACKNOWLEDGEMENT

The authors express their gratitude MAHAJYOTI for supporting to the successful completion of this study.
 
Authors’ contribution
 
Vishal P. Raut: wrote original manuscript, carried out actual experiment and collected data and interpretated results, Manmath D. Sontakke: Conceptualized and validated the study design and reviewed and approved the manuscript. Supriya M. Kamble and Shivam G. Salunke: equally contributed to review the manuscript.
 
Data availability statement
 
The data collected and processed in this study are presented in this published article.
 
Ethics approval and consent to participate
 
The authors affirm that this research study does not require any ethical approval.
 
Consent for publication
 
All authors declare to consent and approve publication of this research.

CONFLICT OF INTEREST

All authors have stated that they do not have any conflicts of interest.

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    Full Research Article

    Nutritional, Sensory and Phytochemical Evaluation of High Protein Granola from Quinoa and Amaranth

    V
    Vishal P. Raut1
    0009-0001-4747-9704
    M
    Manmath D. Sontakke1,*
    0000-0003-1132-8864
    S
    Supriya M. Kamble1
    0009-0009-5038-3293
    S
    Shivam G. Salunke2
    1Department of Food Processing Technology, Institute of Biosciences and Technology, MGM University, Chhatrapati Sambhajinagar-431 001, Maharashtra, India.
    2Sau. K.S.K. (Kaku) College of Food Technology, Beed-431 122, Maharashtra, India.

    ABSTRACT

    Background: The utilization of and plant-derived components is on the rise in the formulation of functional foods that improved nutritional and antioxidant characteristics.

    Methods: Formulations for granola were made using varying level of amaranth, quinoa, foxnut, whey protein isolate, pumpkin seeds and honey. Proximate composition, sensory attributes, antioxidant activity, total phenolic content and, total flavonoid content and color analyses of the samples were determined by the standard analytical methods.

    Result: The granola prepared in this study showed better nutritional and functional qualities than control sample. The protein content of the formulations with whey protein isolate with amaranth was significantly superior, F2 showed 31.2% protein conten and F5 showed the highest energy value (394 kcal). In addition, improved the levels of dietary fibre and minerals like calcium, iron, magnesium, zinc and phosphorus. A color analysis revealed that there were differences between formulations, the ΔE ranged from 1.73 to 7.15. Quinoa, amaranth, pumpkin seeds, foxnut and honey had a significant improvement of antioxidant activity, total phenolic content and the amount of flavonoids. In the formulations, F3 showed the best bioactive and antioxidant effects along with the best mineral composition.

    INTRODUCTION

    Growing consumer awareness of the relationship between diet and health has increased demand for functional foods that provide benefits beyond basic nutrition. Breakfast cereals, especially granola are popular due to their convenience and nutritional values. However many commercial granola are high in sugar and relatively low in protein, creating need for the healthier alternatives (Granato et al., 2020). Commercial cereals are often a source of poor dietary quality and “empty calories” due to their high sugar, salt and saturated fat content and comparatively low dietary fiber, protein and key micronutrient content (Sharma et al., 2020; Singh and Singh, 2019).
           
    The possible health benefits of pseudo cereals unveil that for the preparation of functional food development (Joshi et al., 2019). Examples of pseudo-cereals that have high nutritional quality are amaranth, buckwheat and quinoa. The main reason for the use of quinoa today is its high protein % and quality - the balanced amino acid spectrum with a high lysine and methionine content (Abugoch et al., 2009; Wright et al., 2002;). High in fiber and minerals like calcium and iron (Ando et al., 2002). It is also rich in antioxidants, such as polyphenols (Hirose et al., 2010; Repo-Carrasco-Valencia et al., 2011). Also high protein content in amaranth makes it ideal for use in the preparation of healthful food (gluten-free) for people with gluten hypersensitivity (Capriles et al., 2014). Similarly, Foxnut (Euryale ferox) and pumpkin seeds are rich sources of natural protein (25-37%) and oil (37-45%) antioxidants, minerals and nutrients that contribute to overall heath (Mishra et al., 2022; Kaur et al., 2018; Chauhan et al., 2025). Whey protein isolate (WPI), containing more than 90% protein and all essential amino acids, is widely used to enhance the nutritional quality of food products complementing the plant proteins present in Pseudocereals and seeds (Smithers, 2015). The granola is sweetened with honey and dried fruits/fruit pastes, which are used to increase the energy content of the granola. The honey is natural sweetener, having therapeutic characteristics as well as binder for producing (Deshmukh et al., 2017).
           
    Previous studies have reported the successful incorporation of Pseudocereals and seeds into ceareal- based product, improving their nutritional and functional properties (Rocchetti et al., 2021). However, limited research has investigated the combined use of amaranth, quinoa, Foxnut, pumpkin seeds, honey and whey protein isolate in granola formulations and understanding the complex interactions among ingredients. The combination of these ingredients is expected to create a synergistic effect by improving protein quality, minerals density, antioxidants potential, texture, flavour and overall consumer acceptability. Furthermore, information regarding the physicochemical, textural and evaluate its nutritional, physicochemical, textural and sensory properties.
           
    The research aims to develop gluten-free and nutritious rich Amaranth, Quinoa Granola with the inclusion of Foxnut, Pumpkin seeds with its deliciousness and evaluate its nutritional, physicochemical, textural and sensory properties.

    MATERIALS AND METHODS

    Raw material required for the formulation are Amaranth grain (Amaranthus hypochondriacus), quinoa flour (Chenopodium quinoa), foxnut (Euryale ferox), pumpkin seeds (Cucurbita pepo), honey and whey protein isolate (WPI) of functional granola are procured from local market of Chhatrapati Sambhajinagar, Maharashtra, India. Whey protein isolate (90% protein) was purchased from a commercial manufacturer. Honey was used as the natural sweetener and binder because of its nutritional value and adhesive properties. All the analysis was carried out at Institute of Biosciences and Technology, MGM University Chhatrapati Sambhajinagar, Maharashtra in the year 2024 to 2025.
     
    Formulation of functional granola
     
    Five alternative formulations were built to find the most acceptable sensory properties by varying the amount of amaranth, quinoa and whey protein (Table 1) expressed in % of the total. The 25% honey was uniformly added as a binding agent due to benefits of honey as a texture, cohesion and sensory improver in granola and cereal based product (Sharma and Gujral, 2020; Deshmukh et al., 2017). The proportion of roasted pumpkin seeds and Foxnut was maintained constant in all five formulations. The prepared sample of granola is shown in Fig 1.

    Table 1: Formulation for standardization of functional granola.



    Fig 1: Process of formulated functional granola. (Reference: Ahada et al., 2025).


     
    Proximate analysis
     
    Proximate composition of formulated granola was determined in triplicate (n=3) using standard methods of the AOAC International. Moisture content was determined by the Hot air oven method (AOAC method 925.10), crude fat by Soxhlet extraction, model-soxtron sox-2, crude protein by Kjeldahl method (Kjeltron KGIDB4M, India) using a nitrogen conversion factor of 6.25 (AOAC method 979.09) and total ash by incineration in muffle furnace (AOAC method 923.03) Muffle furnace, Biotechnics India). The total carbohydrate content was calculated by the difference method according to AOAC procedure using the following equation:

    Carbohydrates (%) =
    100 - [Moisture (%) + Protein (%) + Fat (%) + Ash (%)]
     
    Sensory analysis
     
    The sensory evaluation was conducted by 15 semi-trained panellists (aged 22-45 years) familiar with cereals based products. Samples were coded with random three-digit number and served in a randomized order under controlled laboratory conditions. Color and appearance, aroma, taste, body and texture and overall acceptability were evaluated using a nine-point hedonic scale (9= like extremely; 1= dislike extremely). The panel size was considered adequate for preliminary acceptability testing of newly developed food products. Sensory score were analysed using one-way ANOVA followed by Duncan’s multiple range test (DMRT) at p < 0.05 (Lawless et al., 2010).
     
    Shelf-life study: Moisture, pH, acidity and peroxide value
     
    Shelf life of the formulated functional granola was evaluated following the method of Iuliano et al., (2019) did, but with slight modifications. Granola samples were packed in laminated aluminium foil polyethylene pouches and stored at room temperature (30±2oC) under ambient relative humidity conditions for 28 days. Analyses were performed in triplicate at 0, 7, 14, 22 and 28 days of storage. Moisture content (AOAC, 2019), pH, titratable acidity, peroxide value and free fatty acid (FFA) content were determined to access product stability. The 28-day storage period was selected as a preliminary shelf-life assessment to monitor physicochemical changes and oxidative stability of developed granola under normal conditions.
     
    Antioxidant activity, total phenolic content and total flavonoid content
     
    The antioxidant activity, total phenolic content (TPC) and total flavonoid content (TFC) of the formulated granola were determined in triplicate (n=3). Briefly, 1 g of finely ground granola was extracted with 10 mL of methanol (1:10 w/v), vortexes for 5 min and centrifuged at 1400 rpm for 30 min. The supernatant was collected and used for further analyses. Antioxident activity was evaluated using the DPPH radical scavenging assay described by Dubey et al. (2023) and absorbance was measures at 517 nm using a UV-Vis spctrophotmeter [(UV_!*) Shimadzu Corpration, Japan]. The results were expressed as percentage inhibition using the equation:

     
    Where
    A0 = Absorbance of the control.
    As = Absorbance of the sample.
           
    The TPC was determined by Folin-Ciocalteu method using gallic acid as the standard and expressed as mg GAE/100 g sample (Pérez et al., 2023). TFC was determined using the aluminium chloride colorimetric method with quercetin as the standard and expresses as mg QE/100 g sample (Shraim et al., 2021).
     
    Mineral analysis
     
    Calcium, iron, magnesium, zinc and phosphorus content were determined in triplicate according to AOAC (2019). Granola samples were dry-ash at 550oC and the ash was dissolved in dilute hydrochloric acid, filtered and diluted to a known volume. Calcium, iron, magnesium and zinc were quantified using Atomic Absorption Spectrophotometry (AAS) with certified standard solution for calibration, while phosphorus was determined colorimetrically using a standard phosphate calibration curve. Quality control was ensured through reagent blanks, calibration verification standards and duplicate analyses. Mineral concentration was expressed as mg/100 g sample.
     
    Color analysis
     
    The color characteristics of the formulated granola samples were measured using a Hunter Lab MiniScan XE Plus colorimeter. Color values were recorded as L* (lightness), a* (redness/greenness) and b* (yellowness/blueness). The overall color difference (ΔE) between samples was calculated using the CIE Lab* color difference equation (Pathare et al., 2013):.
     
    ΔE = √[(ΔL)2 + (Δa)2 + (Δb*)2]**

    Where 
    ΔL*, Δa* and Δb* = Differences in lightness, redness/greenness and yellowness/blueness, respectively. Measurements were performed in triplicate and results were expressed as mean ± standard deviation.
     
    Statistical analysis

    All experiments were performed in triplicate (n=3) and results were expressed as mean ± standard deviation. Data were analysed using Minitab 20 statistical software. A completely randomized design (CRD) was employed and differences among the six granola formulations (control and five treatment formulation) were evaluated using one-way analysis of variance (ANOVA). Mean comparisons were performed using tukey’s H Honestly Significant Difference (HSD) test at a 95% confidence level (p<0.05). Sensory and storage study data were analysed using the same statistical approach to determine significant differences among formulations and storage periods. 

    RESULTS AND DISCUSSION

    The proximate composition of the formulated granola is presented in Table 2. Protein content increased significantly from 11.8±0.24% in the control to 31.2 ± 0.38% in F2 (p<0.05), mainly due to the addition of whey protein isolate, while amaranth and quinoa provide complementary pant protein (Boire et al., 2025). The slight increase in fat content (5.7-6.8%) was attributed to the healthy unsaturated fats present in pumpkin seeds and Pseudocereals. Crude fibre increased with the incorporation of amaranth , quinoa and Foxnut, with F3 showing the highest value (6.2±0.10%), owing to their naturally high dietary fibre content (Jan et al., 2023) the higher ash content observed in F5 reflected the greater mineral contribution from Pseudocereals and pumpkin seeds, which are rich in calcium, magnesium, iron and zinc (Anuradha et al., 2023; Olawuni et al., 2024).Consequently, carbohydrate content decrease because it was replaced by protein – and fibre-rich ingredient, while the energy value (372-394 kcal/100 g) remained comparable among formulations. These results demonstrate that combined use of Pseudocereals, pumpkin seeds and whey protein isolate enhanced the nutritional quality of granola by improving protein, fibre and mineral contents without substantially increasing energy value.

    Table 2: Nutritional properties of functional granola.


     
    Total phenolic content, antioxidant activity, total flavonoid content
     
    The antioxidant activity, total phenolic content (TPC) and total flavonoid content (TFC) of the granola formulations are presented in Table 3. Formulations enriched with higher proportions of amaranth and quinoa (F3 and F4) exhibited significantly higher antioxidant activity, TPC and TFC due to the abundance of phenolic acid and flavonoids, including quercetin and kampferol, natural present in these Pseudocereals (Alvarez-Jubete et al., 2010).however, not all formulations showed similar improvements. The comparatively lower values observed in F1, F2 and F5 % may be attributed to a dilution effect caused by a higher proportion of ingredients with lower phenolic concentration, as well as possible interactions between protein and phenolic compounds that reduce the extractability of bioactive compounds during analysis (Tang and Tsao, 2017; Zhu, 2025). These findings indicate that the antioxidants potential of granola depends on the composition and interactions among ingredients rather than the addition of functional ingredients alone.

    Table 3: Phytochemical and antioxidant properties of functional granola.


           
    The formulated granola also showed improved mineral composition. Higher calcium, iron, magnesium, zinc and phosphorus content were mainly associated with the incorporation of quinoa, amaranth, pumpkin seeds and whey protein isolate, all of which are naturally rich in essential minerals (Alvarez-Jubete et al., 2010; Aremu et al., 2022:). This demonstrates that combining Pseudocereals with protein-rich ingredients enhances both the bioactive and minerals profiles of the developed granola.
     
    Color analysis  
     
    The color characteristics of the formulated granola are presented in Table 4. The L * values (56.41±0.33-62.68± 0.24) indicates differences in lightness among the formulations, while positive a* values (6.71±0.08-7.72±0.09) and b* values (24.08±0.21-27.55±0.19) confirmed the characteristic reddish-yellow appearance of the granola. These variations were mainly attributed to differences in ingredient composition, particularly the levels of honey, pumpkin seeds, amaranth and quinoa as well as browning reactions during roasting (Szydłowska et al., 2022). The ΔE values indicated perceptible color differences among formulations, suggesting that ingredient substitution produced visually distinguishable products that may influence consumer perception and product acceptability. Overall, the color changes reflected both formulations differences and processing conditions.

    Table 4: Color analysis of functional granola.


     
    Sensory evaluation of functional granola
     
    The sensory scores (Table 5) showed that all formulations were well accepted, with F5 receiving the highest overall acceptability score (H≈9), followed by F1 and F2. The superior acceptability of F5 may be attributed to the balanced combination of amaranth, quinoa, Foxnut, pumpkin seeds, honey and whey protein isolate, which provided a desirable flavour, crisp texture, attractive color and pleasant mouth feel. In contrast, formulations containing higher levels of amaranth (F3 and F4) showed comparatively lower scores, likely due to its characteristic earthy flavour and denser texture, which may have reduced consumer preference (Sharma and Kaur, 2021). The consistent scores obtained from the semi-trained panel indicate good agreements among panellists and support the reliability of the sensory evaluations.

    Table 5: Sensory evaluation of functional granola.


     
    Change in moisture content, pH, acidity, peroxide value
     
    Granola formulations F1-F5 showed consistent moisture stability over the 28-day storage period ranging from about 4.8% to 6.2% moisture. In the samples F2 had a slight decrease from the high moisture content at the beginning and F3 and F4 showed more stable moisture content.
           
    The storage stability of the formulated granola was evaluated over 28 days by monitoring moisture content, pH, titratable acidity and peroxide value (Fig 2). Moisture content showed only a slight change (4.8-6.2%) during storage, indicating that the aluminium foil- polyethylene packaging effectively minimized moisture transfer and maintained produt quality (Fellows, 2009; Robertson, 2016). The pH decreased significantly (p<0.05) from 6.5-6.7 at day 0 to 5.6–6.1 on day 28, reflecting the formation of free fatty acids and other oxidation products during storage (Choe and min, 2006; Zhang et al., 2019).

    Fig 2: Effect of storage on moisture and pH of functional granola.


           
    Titratable acidity increases significantly p < 0.05) in all formulations, from 0.1-0.2% at day 0 to 0.4–0.6% after 28 days. F2 exhibited the highest acidity (0.6%), followed by F3 (0.5%), whereas F1, F4 and F5 reached 0.4%, (showed in Fig 3) suggesting comparatively lower hydrolytic deterioration (Gumus and Decker, 2021). Similarly, peroxide value increased (Fig 3) from 1.8-2.3 to 6.2-6.9 meq O2/kg during storage, indicating progressive lipid oxidation. F4 recorded the highest peroxide value (6.9 meq O‚ /kg), while F3 showed the lowest (6.2 meq O2/kg), demonstrating relatively better oxidative stability (Gumus and Decker, 2021; Shahidi and Zhong, 2015). Although significant physicochemical changes occurred during storage (p< 0.05), all formulations maintained acceptable quality throughout the 28-days storage period under ambient conditions.

    Fig 3: Effect of storage on Acidity and peroxide value of functional granola.


           
    The higher total phenolic and flavonoid content in F3 contributed to better oxidative stability, as reflected by its lower peroxide value (6.2 meq O2/kg) after 28 days. Phenolic compounds act as natural antioxidants by scavenging free radicals and delaying lipid oxidation (Shahidi and Zhong, 2015). In contrast, F3 showed the highest peroxide value (6.9 meq O2 /kg), indicating that oxidative stability depends not only on antioxidant content but also on ingredient composition and interaction among bioactive compounds.

    CONCLUSION

    Overall, the developed Pseudocereals based granola exhibited improved nutritional guilty, with higher protein, fibre, mineral and bioactive compound content while maintaining a comparable energy value. The optimized formulation demonstrated colour, good sensory acceptability and satisfactory oxidative stability, indicating that ingredient composition and interaction significantly influenced product quality. These findings suggest that the combined used of Pseudocereals, pumpkin seeds and whey isolate is an effective approach for developing a nutritious and functional granola with enhanced health-promising properties.

    ACKNOWLEDGEMENT

    The authors express their gratitude MAHAJYOTI for supporting to the successful completion of this study.
     
    Authors’ contribution
     
    Vishal P. Raut: wrote original manuscript, carried out actual experiment and collected data and interpretated results, Manmath D. Sontakke: Conceptualized and validated the study design and reviewed and approved the manuscript. Supriya M. Kamble and Shivam G. Salunke: equally contributed to review the manuscript.
     
    Data availability statement
     
    The data collected and processed in this study are presented in this published article.
     
    Ethics approval and consent to participate
     
    The authors affirm that this research study does not require any ethical approval.
     
    Consent for publication
     
    All authors declare to consent and approve publication of this research.

    CONFLICT OF INTEREST

    All authors have stated that they do not have any conflicts of interest.

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